HU208160B - Process for producing t-butyl-oxycarbonyl-1-tyrosylpeptidoglycan monomer and its derivatives marked with 125 i, as well as pharmaceutical compositions comprising same as active ingredient - Google Patents

Abstract

The present invention relates to the novel tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer
and to its ¹²⁵1-labelled Boc-Tyr-PGM derivative; to the preparation and use thereof in the preparation of pharmaceuticals of immunostimulating and antitumor activity.

Description

Description: 6 pages (including 2 pages)

HU 208 160 B

The present invention relates to a process for the preparation of t-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer and its ¹²⁵ I-labeled derivatives, and to pharmaceutical compositions containing the novel compounds thus prepared. The tert-butyloxycarbonyl-L-tyrosyl peptidoglycan monomer is immunomodulatory and antitumor, and the new ¹²⁵ I derivative has anti-PGM antibody binding activity.

It is known that the peptidoglycan monomer (PGM, GlcNAc-P- (1-4) -MurNAc-L-Ala-D-iGln - [(L) -mesoA ₂ pm- (D) -amide- (L) -D -Ala-D-Ala], which is the smallest repeating unit of the Brevibacterium divaricum cell wall peptidoglycan, exhibits immunostimulatory, antitumor and antimetastatic effects [Tomasic, J. and Hrsak, I. (1988). activities in the heat, in: Surface Structure of Microorganisms and Their Interaction with the Mammalian Heat (Schrinner, E., Richmond, H. H. Seibert, G. and Schwartz, U. Eds.), 113-121, VCH Verlagsgesellschaft , Weinheim)].

It is known that peptide bond formation occurs on the condition that if the amino acid is

a. converting to a N-protected amino acid by introducing a protecting group;

b) activated at the carboxy group;

c) performing an appropriate reaction on the C-terminally protected amino acid, di-, tri- or polypeptide; and

d) deprotection of the resulting di-, tri- or polypeptides by special reactions.

In addition, peptidoglycan monomers containing disaccharide pentapeptides (sugar and peptide moieties) have very complex structure in polyfunctional molecules.

(a) the hydroxyl groups of the GlcNAC and MurNAC units (sugar moieties of the molecule) shall be protected; and

(b) deprotection of the sugar moiety in the peptide bond formation reaction.

It is also known that conventional intake of tyrosine into a peptide or protein is so called. An "active ester process" in which the acylating component is a Boc-L-TyrONSu unit (Assoian, R. K. (1980) Anal. Biochem. 103, 70).

The present invention relates to a tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer (Boc-Tyr-PGM) of formula (I) and its ¹²⁵ I-labeled derivative, which is capable of meeting the growing need for novel immunostimulatory and antitumor activity. .

The novel peptidoglycan monomer of formula (I) allows the preparation of a compound with increased lipophilic properties, which penetrates the cell wall more easily and has a longer residence time. The compound is prepared by introducing the parent aromatic amino acid tyrosine into the PGM molecule to form the disaccharide hexapeptide (Boc-Tyr-PGM) from the disaccharide pentapeptide. Subsequent ¹²⁵ I labeling of the disaccharide hexapeptide gives ¹²⁵ I-Boc-Tyr-PGM derivative II.

The process for preparing the novel peptidoglycan monomer derivative is well known in the art of peptide chemistry; the synthesis is carried out in the fully unprotected state of a molecule of very complex structure.

The new PGM derivatives are prepared according to the following reaction scheme:

ω NH ₂

I Et ³ N Disaccharide Pentapeptide-COOH + Boc-Tyr-ONSu— »1

In the process of the invention, the regio-selective reaction of Boc-Tyr-ONSu and the omegaamino group of the mesodiaminopimelic acid of the PGM molecule in the presence of triethylamine produces a novel disaccharide pentapeptide derivative of a Sephadex G-25 gel chromatography column. after purification by silica gel column chromatography and completely suitable for biological assays of Biogel P-2 gel chromatography.

Radiolabelling of Boc-Tyr-PGM with radioiodine is carried out by the known chloramine T procedure (Bolton, A.E. (1985), in: Radioiodination Techniques, Second Edition, 709, Amersham, Int. Plc, England).

Substitution of an electrophile ortho-ortho to the hydroxyl group of the phenol ring in tyrosine affords the ¹²⁵ I-labeled molecule with high specific activity and appropriate radiochemical stability.

The preparation is carried out according to Scheme 1.

The standard chloramine T procedure for the preparation of the radiolabeled BocTyr-PGM of the present invention using Na [ ²⁵ I] affords an iodinated product of formula II which, after purification by Sephadex G-25 gel chromatography, is antigenic. can be used in antibody binding reactions.

The novel PGM derivative is a disaccharide heptapeptide for use in immunomodulatory and antitumor drugs, while the new ¹²⁵ I-labeled derivative is for use in anti-PGM antibody binding drugs.

The following examples illustrate the invention in more detail.

Example 1

Preparation of tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer (Compound I) 100 mg (0.1 mmol) of peptidoglycan monomer (PGM) were dissolved in 2.6 ml of anhydrous dimethylformamide (DMF). 30 μΐ triethylamine was added. To the cooled (ice water) solution was added 56 ng (0.12 mmol) of Boc-TyrOSu solid (N-hydroxysuccinimide ester of Boc tyrosine) in small portions with stirring. The reaction was allowed to proceed for 16 hours and the DMF was evaporated in vacuo using a mechanical vacuum pump. The thick syrupy evaporation residue was dissolved in water, acidified to pH = 3 with solid citric acid and extracted with ethyl acetate (3 x 10 mL). The aqueous portion was concentrated to 2 mL and filled with water onto a 90 x 2.5 cm Sephadex G-25 column. The 3 mL fractions from the column were subjected to 230 nm absorption, the higher molecular weight fractions were combined and evaporated. The

160 g of the resulting glass residue (110 g) were dissolved in n-butanol: ethanol: 25% (v / v) ammonium hydroxide: water (5: 3: 2: 2) and eluted with the same mixture on a silica gel column. After neutralization of the fractions with HOAc and evaporation, the product was chromatographed on a 70 x 2.5 cm Biogel P-2 column with water. The mobile fractions thus purified were combined, concentrated and lyophilized. 58 ng (46%) of pure Boc-Tyr-PGM (I) monomer were obtained with the following analysis.

GlcNH ₂ 0.82; MurNH ₂ 0.57; Below, 3; Glu, 1; Δ ₂ pm, 0.98; Tyr, 0.80.

1 H NMR (D ₂ O): 1.29 (s, Me ₃ C), 1.34-1.70 (m, partial overlap: Me ₃ C, 21H, 3xMe-Ala + lacto-Me), 1 89 and 1.98 (2s, 6H, 2 x NAc), 6.77 and 7.07 (2d, 2H, I _{H> H,} 8.55 Hz, -C ₆ H ₄ -).

Thin layer chromatography on silica gel:

5: 3: 2: 2 v / v n-butanol: ethanol: 25% v / v ammonium hydroxide: water; detection with iodine vapor and peptide reagent;

R _f = 0.5.

Example 2

Preparation of tert-butyloxycarbonyl [ ¹²⁵ I] -L-tyrosylpeptidoglycan monomer (II) in 5 μg of Boc-Tyr in 25 μΐ (0.5 M, pH 7.5) phosphate buffer For PGM, 10 μΐ (3.7x10 Bq, 1mCi) Na [ ¹²⁵ I] and 50 µg chloramine T (N-chloro-p-toluenesulfonic acid sodium) in 25 μΐ (0.25 M, pH 73) phosphate buffer were added. ) and quenched after 45 seconds by addition of 50 µg of sodium metabisulfite dissolved in 25 µl of water. The reaction mixture was immediately loaded onto a 30 x 1.5 cm Sephadex G-25 column, previously washed with human albumin and equilibrated with 0.025 M pH 7.5 phosphate buffer. The collected 2 ml fractions are subjected to radioactivity per test, and Boc- ^[I25 I] -LTyr-PGM fractions. The resulting derivative II had a specific activity of about 3.12 mBq / pg (0.087 mCi / pg).

Example 3

Effect of tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer (Boc-Tyr-PGM) Immuno-stimulatory effect of Boc-Tyr-PGM was tested in mice immunized with sheep red blood cells (SRBC).

Group CBA or AKR mice were immunized with sheep erythrocytes (1x10 ⁸ SBRC) in Hanks' solution.

After one day (day 1 + 1), the control mice were administered Hanks solution alone, the test groups received 200 pg Boc-Tyr-PGM in Hanks solution, and the positive control group received 200 pg Pancidoglycan monomer (PGM) in Hanks solution. After four days (first day + 4) using the Jeme method [Jerne, N.K., Nordin, A.A. and Henry, C. (1963), The Agar Plaque Technique for Recognizing Antibody-Producing Cells, in: Cell Bound Antibodies, 109, Wistar Institute Press, Philadelphia] determined the number of antibody-forming cells.

The results obtained are summarized in Table 1.

Table 1

Mouse- tribe Treatment PFC value range average value % of average CBA Hanks' solution 95.200 to 238.000 188.906 100 Boc-Tyr-PGM 257.600 to 304.266 296.866 157 PGM 198.800 to 449.866 313.413 166 AKR Hanks' solution 20.000 to 55.200 37.388 100 Boc-Tyr-PGM 31.730 to 68.670 55.974 150 PGM 27.200 to 61.070 48.668 130

Boc-Tyr-PGM shows an immunostimulatory effect in both strains of mice. Effect: 50% increase in PFC count relative to control.

The efficacy of Boc-Tyr-PGF in both mouse strains was comparable to that of the positive control, i.e. the peptidoglycan monomer.

Example 4

Antitumor (Antimetastatic) Effect of Tert-Butyloxycarbonyl-L-Tyrosyl Peptidoglycan Monomer (Boc-Tyr-PGM)

The anti-tumor activity of Boc-Tyr-PGM was performed in mice inoculated with B-16 melanoma. On day 0, 5 groups of 4 month old male C57BL / 6 mice were each given 1x10 ^{3 * 5} B-16 melanoma cells.

The treatment group was no longer given any treatment. Each mouse in the experimental groups was given 1 mg of Boc-Tyr-PGM according to the following protocol.

Group 1: Day 3;

Group 2: Day 7; and

Group 3: Days 3 and 7 (total 2 mg Boc-TyrPGM).

Three PGM (PLIVA) treated groups were used as a positive control group and were treated as above.

Mice were sacrificed at day 23 and lung metastasis was determined macroscopically. The results obtained are summarized in Table 2.

Table 2

Treatment Sun metastasis Inhibition% SzámX % Hanks' solution - 12.4 ± 6.1 100 Boc-Tyr-PGM 3 7.6 ± 1.9 61.3 38.7 7 7.2 ± 4.0 58.1 41.9 3 + 7 7.0 + 2.7 56.4 43.6 PGM (PLIVA) 3 6.2 ± 4.4 50.0 50.0 7 5.4 ± 1.7 43.5 66.5 3 + 7 5.3 ± 1.7 42.7 67.3

HU 208 160 B

Treatment with Boc-Tyr-PGM has a clear anti-tumor effect. As a result of treatment, metastasis was reduced in all treated groups (inhibition rate: 38.7% -43.6%). Treatment with 1 mg at different times (Day 3 or Day 7) and in different amounts (Day 3 or Day 7) or 2 mg total on Days 3 and 7 did not produce any significant difference in inhibition.

The efficacy of Boc-Tyr-PGM is comparable to that of positive control PGM (PLIVA).

Claims (4)

PATENT CLAIMS A process for the preparation of the t-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer of formula (I) and its ¹²⁵ I-labeled derivative, characterized in that tert-butyloxycarbonyl-L-tyrosine-N-hydroxy The succinic imido ester is condensed with a peptidoglycan monomer in the presence of triethylamine, and the resulting tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer is purified by chromatography and, if desired, Na [ ¹²⁵ I] and N are added. The reaction was quenched with sodium bisulfide and isolated by elution with phosphate buffer on a Sephadex G-25 column. 2. A process for the preparation of a pharmaceutical composition comprising the process of claim 1 The tert-butyloxycarbonyl-L-tyrosyl-peptidoglycan monomer prepared is converted into a pharmaceutical composition by admixing with diluents, carriers and / or other excipients customary in the pharmaceutical art. 3. The method of claim 2, wherein the composition is an immunostimulatory drug. The method of claim 2, wherein the antitumor drug is formulated.